The fibroblast growth factor (FGF) family consists of at least eighteen distinct members (Basilico et al., Adv. Cancer Res. 59:115-165, 1992 and Fernig et al., Prog. Growth Factor Res. 5(4):353-377, 1994) which generally act as mitogens for a broad spectrum of cell types. For example, basic FGF (also known as FGF-2) is mitogenic in vitro for endothelial cells, vascular smooth muscle cells, fibroblasts, and generally for cells of mesoderm or neuroectoderm origin, including cardiac and skeletal myocytes (Gospodarowicz et al., J. Cell. Biol. 70:395-405, 1976; Gospodarowicz et al., J. Cell. Biol. 89:568-578, 1981 and Kardami, J. Mol. Cell. Biochem. 92:124-134, 1990). In vivo, bFGF has been shown to play a role in avian cardiac development (Sugi et al., Dev. Biol. 168:567-574, 1995 and Mima et al., Proc. Nat""l. Acad. Sci. 92:467-471, 1995), and to induce coronary collateral development in dogs (Lazarous et al., Circulation 94:1074-1082, 1996). In addition, non-mitogenic activities have been demonstrated for various members of the FGF family. Non-proliferative activities associated with acidic and/or basic FGF include: increased endothelial release of tissue plasminogen activator, stimulation of extracellular matrix synthesis, chemotaxis for endothelial cells, induced expression of fetal contractile genes in cardiomyocytes (Parker et al., J. Clin. Invest. 85:507-514, 1990), and enhanced pituitary hormonal responsiveness (Baird et al., J. Cellular Physiol. 5:101-106, 1987.)
Several members of the FGF family do not have a signal sequence (aFGF, bFGF and possibly FGF-9) and thus would not be expected to be secreted. In addition, several of the FGF family members have the ability to migrate to the cell nucleus (Friesel et al., FASEB 9:919-925, 1995). All the members of the FGF family bind heparin based on structural similarities. Structural homology crosses species, suggesting a conservation of their structure/function relationship (Ornitz et al., J. Biol. Chem. 271(25):15292-15297, 1996.)
There are four known extracellular FGF receptors (FGFRs), and they are all tyrosine kinases. In general, the FGF family members bind to all of the known FGFRs, however, specific FGFs bind to specific receptors with higher degrees of affinity. Another means for specificity within the FGF family is the spatial and temporal expression of the ligands and their receptors during embryogenesis. Evidence suggests that the FGFs most likely act only in autocrine and/or paracrine manner, due to their heparin binding affinity, which limits their diffusion from the site of release (Flaumenhaft et al., J. Cell. Biol. 111(4) :1651-1659, 1990.) Basic FGF lacks a signal sequence, and is therefore restricted to paracrine or autocrine modes of action. It has been postulated that basic FGF is stored intracellularly and released upon tissue damage. Basic FGF has been shown to have two receptor binding regions that are distinct from the heparin binding site (Abraham et al.,. EMBO J. 5(10):2523-2528, 1986.)
It has been shown that FGFR-3 plays a role in bone growth. Mice made homozygous null for the FGFR-3 (xe2x88x92/xe2x88x92) resulted in postnatal skeletal abnormalities (Colvin et al., Nature Genet. 12:309-397, 1996 and Deng et al., Cell 84:911-921, 1996). The mutant phenotype suggests that in normal mice, FGFR-3 plays a role in regulation of chrondrocyte cell division in the growth plate region of the bone (Goldfarb, Cytokine and Growth Factor Rev. 7(4) :311-325, 1996). The ligand for the FGFR-3 in the bone growth plate has not been identified.
Although four FGFRs have been identified, all of which have been shown to have functional splice variants, the possibility that novel FGF receptors exist is quite likely. For example, no receptor has been identified for the FGF-8a isoform (MacArthur et al., J. Virol. 69(4) :2501-2507, 1995.).
FGF-8 is a member of the FGF family that was originally isolated from mammary carcinoma cells as an androgen-inducible mitogen. It has been mapped to human chromosome 10q25-q26 (White et al., Genomics 30:109-11, 1995.) FGF-8 is involved in embryonic limb development (Vogel et al., Development 122:1737-1750, 1996 and Tanaka et al., Current Biology 5(6) :594-597, 1995.) Expression of FGF-8 during embryogenesis in cardiac, urogenital and neural tissue indicates that it may play a role in development of these tissues (Crossley et al., Development 121:439-451, 1995.) There is some evidence that acrocephalosyndactylia, a congenital condition marked by peaked head and webbed fingers and toes, is associated with FGF-8 point mutations (White et al., 1995, ibid.)
FGF-8 has five exons, in contrast to the other known FGFs, which have only three exons. The first three exons of FGF-8 correspond to the first exon of the other FGFs (MacArthur et al., Development 121:3603-3613, 1995.) The human gene for FGF-8 codes for four isoforms which differ in their N-terminal regions: FGF isoforms a, b, e, and f; in contrast to the murine gene which gives rise to eight FGF-8 isoforms (Crossley et al., 1995, ibid.) Human FGF-8a and FGF-8b have 100% homology to the murine proteins, and FGF-8e and FGF-8f proteins are 98% homologous between human and mouse (Gemel et al., Genomics 35:253-257, 1996.)
Heart disease is the major cause of death in the United States, accounting for up to 30% of all deaths. Myocardial infarction (MI) accounts for 750,000 hospital admissions per year in the U.S., with more than 5 million people diagnosed with coronary disease. Risk factors for MI include diabetes mellitus, hypertension, truncal obesity, smoking, high levels of low density lipoprotein in the plasma or genetic predisposition.
Cardiac hyperplasia is an increase in cardiac myocyte proliferation, and has been demonstrated to occur with normal aging in the human and rat (Olivetti et al., J. Am. Coll. Cardiol. 24(1):140-9, 1994 and Anversa et al., Circ. Res. 67:871-885, 1990), and in catecholamine-induced cardiomyopathy in rats (Deisher et al., Am. J. Cardiovasc. Pathol. 5(l1):79-88, 1994.) Whether the increase in myocytes originate with some progenitor cell, or are a result of proliferation of a more terminally differentiated cell type, remains controversial.
However, because infarction and other causes of myocardial necrosis appear to be irreparable, it appears that the normal mechanisms of cardiac hyperplasia cannot compensate for extensive myocyte death, and there remains a need for exogenous factors that promote hyperplasia and ultimately result in renewal of the heart""s ability to function.
Bone remodeling is the dynamic process by which tissue mass and skeletal architecture are maintained. The process is a balance between bone resorption and bone formation, with two cell types thought to be the major players. These cells are the osteoblast and osteoclast. Osteoblasts synthesize and deposit matrix to become new bone. The activities of osteoblasts and osteoclasts are regulated by many factors, systemic and local, including growth factors.
While the interaction between local and systemic factors has not been completely elucidated, there does appear to be consensus that growth factors play a key role in the regulation of both normal skeletal remodeling and fracture repair.. Some of the growth factors that have been identified in bone include: IGF-I, IGF-II, TGF-xcex21, TGF-xcex22, bFGF, aFGF, PDGF and the family of bone morphogenic proteins (Baylink et al., J. Bone Mineral Res. 8 (Supp. 2):S565-S572, 1993).
When bone resorption exceeds bone formation, a net loss in bone results, and the propensity for fractures is increased. Decreased bone formation is associated with aging and certain pathological states. In the U.S. alone, there are approximately 1.5 million fractures annually that are attributed to osteoporosis. The impact of these fractures on the quality of the patient""s life is immense. Associated costs to the health care system in the U.S. are estimated to be $5-$10 billion annually, excluding long-term care costs.
Other therapeutic applications for growth factors influencing bone remodeling include, for example, the treatment of injuries which require the proliferation of osteoblasts to heal, such as fractures, as well as stimulation of mesenchymal cell proliferation and the synthesis of intramembraneous bone which have been indicated as aspects of fracture repair (Joyce et al. 36th Annual Meeting, Orthopaedic Research Society, Feb. 5-8, 1990. New Orleans, La.).
The present invention provides such polypeptides for these and other uses that should be apparent to those skilled in the art from the teachings herein.
The present invention provides an isolated polynucleotide molecule encoding a fibroblast growth factor (FGF) homolog comprising a polynucleotide sequence that encodes for a polypeptide that is at least 80% identical to the amino acid sequence as shown in SEQ ID NO: 2 from amino acid residues 55 to 175.
In other embodiments, the polynucleotide molecule encodes for a polypeptide that is at least 80% identical residues 55 to 196 or 207 of SEQ ID NO: 2.
In another aspect, the present invention provides for an isolated polynucleotide molecule comprising a polynucleotide sequence encoding for a polypeptide that is at least 60% identical to the amino acid sequence as shown in SEQ ID NO: 2 from amino acid residues 28 to 175.
In other embodiments, the polynucleotide molecule encodes for a polypeptide that is at least 80% or 90% identical to the amino acid sequence as shown in SEQ ID NO: 2 from amino acid residues 28 to 175.
In another aspect, the present invention provides for an isolated polynucleotide molecule comprising a polynucleotide sequence encoding for a polypeptide that is at least 60% identical to the amino acid sequence as shown in SEQ ID NO: 2 from amino acid residues 28 to 196.
In other embodiments, the polynucleotide molecule encodes for a polypeptide that is at least 80% or 90% identical to the amino acid sequence as shown in SEQ ID NO: 2 from amino acid residues 28 to 196.
In another aspect, the present invention provides for an isolated polynucleotide molecule comprising a polynucleotide sequence encoding for a polypeptide that is at least 60% identical to the amino acid sequence as shown in SEQ ID NO: 2 from amino acid residues 28 to 207.
In other embodiments, the polynucleotide molecule encodes for a polypeptide that is at least 80% or 90% identical to the amino acid sequence as shown in SEQ ID NO: 2 from amino acid residues 28 to 207.
In other aspects, the present invention provides for a polynucleotide molecule as shown in SEQ ID NO: 1 or SEQ ID NO: 6 from nucleotide 163 or 82 to nucleotide 525 or 585.
In other aspects, the present invention provides expression vectors and cultured cells containing DNA segments comprising the polynucleotide molecules encoding for the FGF homolog polypeptides.
In another aspect, the present invention provides for a method of producing an FGF homolog, wherein the cultured cell expresses polypeptide encoded by polynucleotides comprising the sequences disclosed herein.
In another aspect, the present invention provides an isolated FGF homolog polypeptide comprising an amino acid sequence that is at least 80% identical to the sequence as shown in SEQ ID NO: 2 from residues 55 to 175.
In another aspect, the present invention provides for a polypeptide that is at least 60% identical to the sequence of amino acid residues as shown in SEQ ID NO: 2 from residues 28 to 175.
In other embodiments, the polypeptides are at least 80% or 90% identical to the sequence as shown in SEQ ID NO: 2 from residues 28 to 175.
In another aspect, the present invention provides for a polypeptide that is at least 60% identical to the sequence of amino acid residues as shown in SEQ ID NO: 2 from residues 28 to 196.
In other embodiments, the polypeptides are at least 80% or 90i identical to the sequence as shown in SEQ ID NO: 2 from residues 28 to 196.
In another aspect, the present invention provides for a polypeptide that is at least 60% identical to the sequence of amino acid residues as shown in SEQ ID NO: 2 from residues 28 to 207.
In other embodiments, the polypeptides are at least 80% or 90% identical to the sequence as shown in SEQ ID NO: 2 from residues 28 to 207.
In another aspect, the present invention provides for pharmaceutical compositions of the FGF homolog polypeptides in combination with a pharmaceutically acceptable vehicle.
In another aspect, the present invention provides for an FGF homolog fusion protein with a first and second portion joined by a peptide bond.
In another aspect, the present invention provides for a method expanding mesenchymal cells population comprising administering an FGF homolog polypeptide as shown in SEQ ID NO: 2 from residues 28 to 175, wherein the polypeptide increases the number of cells as compared to cell populations without the polypeptide.
In other embodiments, the cells are cardiac myocytes, skeletal myocytes, fibroblasts, osteoblasts and pluripotent stem cells.
In another aspect, the present invention provides for a method for improving cardiac performance in a patient by administering a therapeutic amount of an FGF homolog polypeptide as shown in SEQ ID NO: 2 from residue 28 to residue 175 and results in improvement in cardiac performance.
In other embodiments, the cardiac improvement is measure as increase in total ejection fraction, a decrease in end diastolic pressure, an increase in dP/dt or a decrease in vascular resistance.
In other aspect, the present invention provides for increasing cardiac performance in an individual comprising administering an effective amount of a composition comprising an FGF homolog polypeptide as shown in SEQ ID NO: 2 from residue 28 to residue 175, where the composition results in improved cardiac performance.